Nitrogen regulator GlnR directly controls transcription of prpDBC operon involved in methylcitrate cycle in Mycobacterium smegmatis .

Mycobacterium tuberculosis utilizes fatty acids of the host as the carbon source. Metabolism of odd chain fatty acids by Mycobacterium tuberculosis produces propionyl-CoA. Methylcitrate cycle is essential for Mycobacteria to utilize the propionyl-CoA to persist and grow on these fatty acids. In M. smegmatis , methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in methylcitrate cycle are encoded by prpC, prpD, and prpB, respectively, in operon prpDBC In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region of prpDBC operon and inhibits its transcription. The binding motif of GlnR was identified by bioinformatic analysis and validated using DNase I footprinting and electrophoretic mobility shift assays. The GlnR-binding motif is seperated by a 164-bp sequence from the binding site of PrpR, a pathway-specific transcriptional activator of methylcitrate cycle, but the binding affinity of GlnR to prpDBC is much stronger than that of PrpR. Deletion of glnR resulted in faster growth in propionate or cholesterol medium compared to wild-type strain. The Δ glnR mutant strain also showed a higher survival rate in macrophages. These results illustrated that the nitrogen regulator GlnR regulates methylcitrate cycle through direct repression of the transcription of prpDBC operon. The finding not only suggests an unprecedented link between nitrogen metabolism and methylcitrate pathway, but also reveals a potential target for controlling the growth of pathogenic mycobacteria. IMPORTANCE The success of mycobacteria survival in macrophage depends on its ability to assimilate fatty acids and cholesterol from the host. The cholesterol and fatty acids are catabolized via β-oxidation to generate propionyl-CoA, which is then primarily metabolized via the methylcitrate cycle. Here, we found a typical GlnR binding box in the prp operon, and the affinity is much stronger than that of PrpR, a transcriptional activator of methylcitrate cycle. Furthermore, GlnR repressed the transcription of prp operon. Deletion of glnR significantly enhanced the growth of Mycobacterium tuberculosis in propionate or cholesterol medium, as well as viability in macrophages. These findings provide new insights into the regulatory mechanisms underlying the crosstalk of nitrogen and carbon metabolisms in mycobacteria.